Plasma high density lipoprotein (HDL) is the principal carrier of plasma cholesterol for biliary cholesterol secretion. The HDL receptor Scavenger Receptor B-l (SR-BI) mediates the uptake of HDL cholesterol and cholesteryl ester for excretion into bile, and its expression correlates with biliary cholesterol secretion. SR-BI knockout mice have a significant decrease in biliary cholesterol secretion, indicating an important role of SR-BI in that process. However, little is known regarding the molecular mechanisms by which SR-BI mediates hepatobiliary secretion of cholesterol. SR-BI is expressed on both sinusoidal and canalicular membranes in liver, and undergoes transcytosisto the canalicular membrane. The sorting signals necessary for SR-BI basolateral targeting and transcytosis to the canalicular membrane are unknown. We have defined three important factors that may regulate SR-BI subcellular sorting in liver and thus SR-BI- dependent biliary cholesterol secretion. One factor is PDZK1, a PDZ domain protein that has been shown to interact with SR-BI and to be essential to maintain hepatic SR-BI levels. A second factor is a basolateral targeting signal in the C-terminus of SR-BI, and a third factor is a cholesterol binding domain in the C- terminal transmembrane domain of SR-BI. The C-terminal transmembrane domain of SR-BI can directly bind cholesterol but is not important for SR-BI-mediated selective uptake or cholesterol efflux. Cholesterol binding by SR-BI in liver may play a role in SR-BI transcytosis. Specific Aim 1 will determine the role of PDZK1 in SR-BI sorting. We have generated a PDZK1 knockout mouse model and will use both PDZK1 deficient mice and in vitro polarized cells models to examine the role of PDZK1 in SR-BI sorting. Specific Aim 2 will delimit the basolateral targeting signal of SR-BI and test the role of this sequence in SR-BI sorting in vivo and in vitro as well as in biliary cholesterol secretion in vivo. Specific Aim 3 will test the hypothesis that the SR-BI cholesterol binding domain plays a role in SR-BI transcytosis. We will test the role of this domain in biliary cholesterol secretion in vivo and SR-BI transcytosis in vitro. These studies will provide fundamental insights into the mechanisms regulating SR-BI sorting and biliary cholesterol secretion.